Atypical cholangiocytes derived from hepatocyte-cholangiocyte transdifferentiation mediated by COX-2: a kind of misguided liver regeneration

Background Hepatocyte-cholangiocyte transdifferentiation (HCT) is a potential origin of proliferating cholangiocytes in liver regeneration after chronic injury. This study aimed to determine HCT after chronic liver injury, verify the impacts of HCT on liver repair, and avoid harmful regeneration by understanding the mechanism. Methods A thioacetamide (TAA)-induced liver injury model was established in wild-type (WT-TAA group) and COX-2 panknockout (KO-TAA group) mice. HCT was identified by costaining of hepatocyte and cholangiocyte markers in vivo and in isolated mouse hepatocytes in vitro. The biliary tract was injected with ink and visualized by whole liver optical clearing. Serum and liver bile acid (BA) concentrations were measured. Either a COX-2 selective inhibitor or a β-catenin pathway inhibitor was administered in vitro. Results Intrahepatic ductular reaction was associated with COX-2 upregulation in chronic liver injury. Immunofluorescence and RNA sequencing indicated that atypical cholangiocytes were characterized by an intermediate genetic phenotype between hepatocytes and cholangiocytes and might be derived from hepatocytes. The structure of the biliary system was impaired, and BA metabolism was dysregulated by HCT, which was mediated by the TGF-β/β-catenin signaling pathway. Genetic deletion or pharmaceutical inhibition of COX-2 significantly reduced HCT in vivo. The COX-2 selective inhibitor etoricoxib suppressed HCT through the TGF-β-TGFBR1-β-catenin pathway in vitro. Conclusions Atypical cholangiocytes can be derived from HCT, which forms a secondary strike by maldevelopment of the bile drainage system and BA homeostasis disequilibrium during chronic liver injury. Inhibition of COX-2 could ameliorate HCT through the COX-2-TGF-β-TGFBR1-β-catenin pathway and improve liver function. Supplementary Information The online version contains supplementary material available at 10.1186/s41232-023-00284-4.


Isolation of mouse hepatocytes
Primary hepatocytes were isolated from mice in the WT-NS, WT-TAA, and KO-TAA groups using a previously described method. 1 In general, mice were anesthetized and the portal vein was cannulated. Then, the liver was perfused and digested with warm collagenase (Sigma-Aldrich, #C9891) solution. Digestion was stopped once cracks appeared on the surface of the liver. Next, the liver was excised and placed in a 10 cm disk containing cold isolation buffer. The liver was then torn apart with forceps and rinsed to release liver cells into the isolation buffer. The cell suspension was collected and filtered through a 70 μm pore size nylon strainer (Nest, #258368) and centrifuged at 150×g for 2 minutes. After centrifugation, the supernatant was discarded and the pellet was resuspended in 50% Percoll solution (Solarbio, #P8370), followed by density gradient centrifugation at 150×g for 10 minutes. Afterward, the pellets were washed with a culture medium and the viability of the yielded cells was measured by trypan blue staining. Cells were plated and cultured overnight before they became attached and showed a cuboidal shape and discernable nuclei.

Histology, immunohistochemistry (IHC), and immunofluorescence (IF)
Liver tissue was fixed in 4% paraformaldehyde and embedded in paraffin. Liver sections (4 μm) were stained with hematoxylin and eosin (H&E) following standard protocols. Detection of the cholangiocyte marker cytoskeleton keratin CK19 was performed. Liver sections were deparaffinized and rehydrated before antigen retrieval using sodium citrate buffer. Then, sections were incubated in 3% H2O2 for 15 minutes and blocked for 1 hour. After that, sections were incubated with primary antibodies overnight at 4 ℃ followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 30 minutes. Finally, sections were stained with 3, 3ʹdiaminobenzidine (DAB) solution and counter-stained with hematoxylin.
For immunofluorescence, liver sections were deparaffinized, rehydrated, and antigen retrieved as described above. Cells, they were seeded on glass chamber slides and fixed with 4% paraformaldehyde for 15 minutes. The slides were then permeabilized with 0.5% Triton X-100 for 15 minutes and blocked for 1 hour. After incubation with primary antibodies, sections were incubated with fluorochromeconjugated secondary antibodies in the dark for 1 hour. Finally, sections were coverslipped using antifade reagents with DAPI and visualized using a fluorescence microscope (Olympus, EX53) or a two-photon confocal microscope (Nikon A1R MP+).
Three to five fields at 100× magnification were randomly selected for each section and semi-quantitative analysis. All images were analyzed by ImageJ software. All primary antibodies used are listed in Supplementary Table S2.

RNA extraction and quantitative real-time PCR
RNA was extracted from 20 mg of the frozen liver using a commercially available kit (Frogene, #RE-03011). 7 μL of RNA was reverse-transcribed using Revertaid first strand cDNA synthesis kit (Thermofisher, #K1622). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Mix. The expression level of mRNA was determined by a CFX96 real-time PCR detection system (Bio-Rad) using the 2 −ΔΔCt method and shown as fold changes. Primer sequences are listed in Supplementary Table   S3.

Transmission Electron Microscopy (TEM)
For transmission electron microscopy, the livers were perfused with normal saline, excised, and fixed in 2.5% glutaraldehyde (Solarbio, #P1126) overnight at 4℃. The samples were then washed with PBS and fixed with 1% osmium tetroxide solution before being dehydrated and embedded in araldite resin. Subsequently, 70-90 nm sections were stained with lead citrate and uranyl acetate and then imaged by TEM (H-600IV Hitachi, Tokyo, Japan).

Protein extraction and Western blot
Frozen liver tissues and cells were homogenized, and total proteins were extracted using a protein extraction kit (KeyGen Biotech, #KGBSP002). Equal amounts (50 μg for tissues and 30 μg for cells) of each sample were loaded onto an SDS-PAGE gel and separated before being transferred to PVDF membranes (Merck Millipore, #IPVH00010). Then, the membranes were blocked with 5% non-fat milk and incubated with primary antibodies overnight at 4 ℃. After incubation with horseradish peroxidase-conjugated secondary antibodies, protein bands were visualized using BeyoECL Star reagent (Beyotime, #P0018AM). The protein level was determined using ImageJ software and shown as relative expression to GAPDH. All primary antibodies used are listed in Supplementary Table S2.

Bile acid measurement
The concentrations of different bile acid species in liver tissue and serum were measured by Metware Biotechnology Inc (Wuhan, China). In general, 20 mg (± 1 mg) of liver and 50 μL of serum collected from each group of mice were added to an internal standard and a steel ball, and then 200 μL of methanol was added to the homogenate.
Samples were shaken at 2500 rpm for 10 minutes and then kept in a -20℃ refrigerator for 10 minutes before being centrifuged at 12000 rpm for 10 minutes. After that, the supernatant was concentrated in the concentrator and reconstituted with 100 μL of 50%

mRNA sequencing and data analysis
Mouse liver tissues and primary mouse hepatocytes were collected, and transcriptome sequencing was performed by Novogene Co., Ltd (Beijing, China).
Generally, RNA was extracted from liver tissues and cells, and a library was obtained for transcriptome sequencing. After the library was qualified, the different libraries are pooling according to the effective concentration and the target amount of data off the machine, and then sequenced by the Illumina NovaSeq 6000. The FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of two conditions/groups (three biological replicates per condition) was performed using the DESeq2 R package (1.20.0). The resulting p-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. padj<=0.05 and |log2(fold change)| >= 1 were set as the thresholds for significantly differential expression. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package (3.8.1). GO terms with corrected p-values less than 0.05 were considered significantly enriched by differentially expressed genes.
ClusterProfiler R package (3.8.1) was used to test the statistical enrichment of differential expression genes in KEGG pathways. Reactome pathways with corrected p-values less than 0.05 were considered significantly enriched by differentially expressed genes. The raw data of mRNA-sequencing have been deposited in the NCBI Gene Expression Omnibus.

Liver metabolomic assay
The liver metabolomic assay was performed by Metware Biotechnology Inc. Generally, samples were thawed on ice, and hydrophilic and hydrophobic compounds were extracted. The sample extracts were analyzed using an LC-ESI-MS

Retrograde ink injection and optical liver clearing
Mice were anesthetized and subjected to laparotomy, and the common bile duct was ligated on the distal side. Carbon black ink was slowly infused into the gallbladder using a 36G needle under a stereomicroscope. The injection was stopped when black dots appeared on the surface of the liver. Then, the whole liver was excised and fixed in 4% paraformaldehyde for 1 day. Afterward, the liver was washed in PBS, soaked in the Tissue-Clearing Reagent CUBIC-L (TCI, T3740), and gently shaken at 37℃ for 1 week for delipidation. Finally, the liver was soaked in Tissue-Clearing Reagent CUBIC-R+(M) (TCI, T3741) and gently shaken at 37℃ for 1 week for refractive index matching. After the optical clearing process, the biliary tree was visualized and observed using a stereomicroscope. The liver was sectioned after ink injection and fixation, and IF was performed using the same protocol as above.

Public data mining
JASPAR is an open-access database of curated and non-redundant transcription factor binding profiles (https://jaspar.genereg.net/). The JASPAR database was applied to predict the regulatory effect of TCF4 on the gene expression of hepatocyte and cholangiocyte makers. Two datasets were used (MA0830.1 and MA0830.2).